Current treatment options for glioblastoma (GB) patients are limited and largely ineffective. The highly invasive nature of GB instills a high resistance to standard therapies and recurrence is virtually assured. The development of new therapeutic strategies coupled with increased selectivity of the treatments is essential for the management and enhanced survival of GB patients. This project brings together an existing team with expertise in biological target validation, high-throughput assay development, and molecular chemical libraries to target the TROY signaling pathway(s) as an innovative approach to treat GB invasion. We have developed cell-based assays for high throughput screening (HTS) to identify small molecule inhibitors of TROY dependent signaling. Our cell-based assay will interrogate both orthotopic and allosteric modulators that have functional consequences for TROY signaling. We will utilize resources available at the Prebys Center at Sanford- Burnham to perform a cell-based HTS in 1536-well format of the NIH's structurally diverse chemical library to identify drug-like compounds that inhibit the TROY signaling pathway. Selectivity profiles will be made using in- hand cell-based counterscreen assays. Hits will be further evaluated for biological effects in TROY-dependent glioma cell lines while not affecting TROY negative cells. Mechanism of action (MOA) studies will assess the impact of validated hits on the disruption of TROY oligomerization, TROY-TRAF2 interaction, and TROY- dependent intracellular signaling pathways. Finally, structure-activity relationship (SAR) by purchase of analogs of validated hit series will be performed along with supporting pharmacological characterization on the best available probe(s). Studies will emphasize the identification of high affinity small molecules with appropriate physicochemical properties to promote passage across the blood-brain-barrier (BBB) and delivery into the brain parenchyma. To capitalize on the strong biology/chemistry synergy among the assembled team of investigators with a goal to identify research tool compounds targeting TROY for future hit-to-lead optimization campaigns, the following Specific Aims are proposed: Aim 1: Implement a cell-based reporter assay and perform a HTS of the NIH MLSMR >365,000 collection for modulators of the TROY signaling pathway; Aim 2: Confirm initial activity and validate hits for potency and selectivity/non-cytotoxicity with existing secondary cellular assays for TROY pathway dependence and general cytotoxicity; Aim 3: Perform a limited structure-activity relationship (SAR) elucidation through analogs by catalogs through the hit validation cascade of secondary assays to select the most potent and selective chemical probe; Aim 4: Perform functional biological assays and tertiary assays on validated scaffolds to elucidate the potential mechanisms of action and efficacy in inhibition of cellular migration, invasion, and resistance in clinically relevant primary GB cells; Aim 5: Perform limited ADME/T and in vitro BBB profiling on the most promising tractable probe(s) and scale up (25 - 50 mg) for a limited rodent PK study and future proof of concept and research studies.